This invention relates to newly identified polynucleotides, polypeptides encoded by certain of these polynucleotides, molecular complexes of RNAs and polypeptides, the uses of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides and recombinant host cells transformed with the polynucleotides. The invention relates particularly to such polynucleotides and polypeptides from Staphylococci, especially S. aureus. This invention also relates to inhibiting the biosynthesis, assembly or action of such polynucleotides and/or polypeptides and to the use of such inhibitors in therapy.
This invention relates to a novel bacterial ribonucleoprotein complex and the component parts thereof. More specifically, this invention relates to RNase P, particularly RNase P from Staphylococcus aureus, and the use of RNase P or components thereof in screens for the identification of antimicrobial compounds and to the use of such compounds in therapy.
The Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
The frequency of Staphylococcus aureus infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
While certain Staphylococcal proteins associated with pathogenicity have been identified, e.g., coagulase, hemolysins, leucocidins and exo- and enterotoxins, additional targets are always useful because it is appreciated that the target of a antimicrobial screen can often bias the outcome. Thus, new targets allow for the discovery of new classes of antimicrobials.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of RNaseP and polypeptides encoded thereby.
In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing RNaseP expression, treating disease, for example, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis), assaying genetic variation, and administering a RNaseP polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus aureus bacteria.
In certain preferred embodiments of the invention there are provided antibodies against RNaseP polypeptides.
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
In accordance with yet another aspect of the invention, there are provided RNaseP agonists and antagonists, preferably bacteriostatic or bacteriocidal agonists and antagonists.
A preferred embodiment of the invention provides an antagonist that inhibits or an agonist that activates an activity of an RNA selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate;
Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kj=8=1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Further provided is a method for the treatment of an individual having need to inhibit or activate RNAseP RNA or holoenzyme comprising the steps of: administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agonist that activates an activity of a RNA selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 MM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211 -213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Still further provided is a method for the treatment of an individual infected with a bacteria comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agonist that activates an activity of a RNA selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Preferred methods are also provided wherein said bacteria is selected from the group consisting of: a member of the genus Staphylococcus, Staphylococcus aureus, a member of the genus Streptococcus, and Streptococcus pneumoniae. 
Another preferred embodiment of the invention is a method for the treatment of an individual having need to inhibit or activate RNaseP RNA or holoenzyme comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agoinist that activates an activity of RNaseP RNA or holoenzyme selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
A further provided embodiment of the invention provides a method for the treatment of an individual infected with a bacteria comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agonist that activates that activates an activity of RNaseP selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A 169, 292-294 (L15.2), 271(bulged A).
A further method for the treatment of an individual infected by Streptococcus pneumoniae is provided comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or anagonist that activates an activity of Streptococcus pneumoniae RNAseP RNA or holoenzyme selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Still further provided is an antagonist that inhibits an activity of a polypeptide selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Also provided is a method for the treatment of an individual having need to inhibit RNaseP RNA or holoenzyme comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits an activity of a polypeptide selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Another preferred embodiment of the invention provides a method for inhibiting an activity of RNAseP RNA or holoenzyme comprising the steps of contacting a composition comprising said polypeptide with an effective amount of an antagonist that inhibits an activity of RNAseP, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc. 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211 -213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
The invention provides a method for inhibiting an activity of RnaseP RNA or holoenzyme, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP KM=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Also provided is a method for inhibiting a growth of bacteria comprising the steps of contacting a composition comprising bacteria with an antibacterially effective amount of an antagonist that inhibits an activity of RNAseP RNA or holoenzyme, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
Another embodiment of the invention is a method for inhibiting a RNaseP RNA or holoenzyme comprising the steps of contacting a composition comprising bacteria with an antibacterially effective amount of an antagonist that inhibits an activity of RNaseP, wherein said activity is selected from the group consisting of: Staphylococcus aureus RNaseP Km=53xc2x14 nM determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate; Staphylococcus aureus RNaseP kcat=3.4xc2x10.1 minxe2x88x921 determined in a reaction in 1xc3x97buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe F as a substrate; Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8xc2x11 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH,OAc, 10 mM MgCl2, and 5% glycererol; Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A 169, 292-294 (L15.2), 271(bulged A).
In a further aspect of the invention there are provided compositions comprising a RNaseP polynucleotide or a RNaseP polypeptide for administration to a cell or to a multicellular organism.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.